The Grapes checkpoint coordinates nuclear envelope breakdown and chromosome condensation.

Mutations in the embryonic Drosophila Grapes/Chk1 checkpoint result in an abbreviated interphase, chromosome condensation defects and metaphase delays. To clarify the relationship between these phenotypes, we simultaneously timed multiple nuclear and cytoplasmic events in mutant grp-derived embryos. These studies support a model in which grp disrupts an S-phase checkpoint, which results in progression into metaphase with incompletely replicated chromosomes. We also show that chromosome condensation is independent of the state of DNA replication in the early embryo. Therefore, grp condensation defects are not a direct consequence of entering metaphase with incompletely replicated chromosomes. Rather, initiation of chromosome condensation (ICC) occurs at the normal time in grp-derived embryos, but the shortened interval between ICC and metaphase does not provide sufficient time to complete condensation. Our results suggest that these condensation defects, rather than incomplete DNA replication, are responsible for the extensive metaphase delays observed in grp-derived embryos. This analysis provides an example of how the loss of a checkpoint can disrupt the timing of multiple events not directly monitored by that checkpoint. These results are likely to apply to vertebrate cells and suggest new strategies for destroying checkpoint-compromised cancer cells.

Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes.

Immunoelectron microscopy with protein A gold has been used to determine the subcellular location of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. RESA was associated with dense vesicles presumed to be micronemes within merozoites. RESA was not detected on the surface of merozoites but was located at the membrane of erythrocytes infected with ring-stage parasites. RESA within merozoites was largely soluble in the nonionic detergent Triton X-100, but was insoluble in this detergent when associated with the erythrocyte membrane.

Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.

Expression of Schistosoma japonicum antigens in Escherichia coli.

A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase. Colonies expressing the S. japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms. In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera. In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria. Several major S. japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.

Expression of Taenia taeniaeformis antigens in Escherichia coli.

Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations. Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies. To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted. Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3. Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein. Four clones that reacted with antibodies in an E. coli colony immunoassay were selected for detailed characterization. Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum. Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens. The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000. This approach allows the assessment of antigens expressed in E. coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite.

A method which facilitates the ordering of DNA restriction fragments.

The Southern transfer technique has been used to provide a generally applicable method for ordering DNA restriction fragments. It involves electrophoresis of partially digested DNA, transfer to nitrocellulose filter paper and annealing to a 32P-labelled fragment. Only those partials containing that particular fragment will reanneal to the probe and produce bands on autoradiography. The size of each partial in the labelled set is the sum of the sizes of the fragment used as probe and of one or more adjacent fragments. Thus the size of the adjacent fragments can be determined from the size increments of this set of partials. The method is illustrated by the mapping of certain BamHI sites on coliphage 186 DNA.

Restriction cleavage maps of coliphages 186 and P2.

The restriction enzymes BamHI, Bg/II, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, Bg/II-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and Xho-I-1; b) P2, BamHI-3, Bg/II-2, EcoRI-3, HindIII-0, PstI-O, XbaI-1 and XhoI-O. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.

Immunochemical analysis of Taenia taeniaeformis antigens expressed in Escherichia coli.

Previously we reported the isolation of several Escherichia coli clones expressing fragments of Taenia taeniaeformis antigens as beta-galactosidase fused proteins (Bowtell, Saint, Rickard & Mitchell, 1984). Here we describe the isolation of additional antigen-expressing clones from a larval cDNA library and the assignment of these clones to 7 antigen families. These were isolated with a polyspecific rabbit antiserum raised to the oncosphere. Since this serum was capable of reacting with a large number of antigens, it was important to develop techniques for rapidly determining the identity of the native T. taeniaeformis molecule corresponding to a cloned antigen gene. These included active immunization of rabbits with fused proteins and several techniques involving affinity purification on immobilized fused proteins. The reactivity of the antigen-positive clones with sera from humans infected with related parasites was also assessed. Finally, immunization of mice with several fused proteins failed to protect against subsequent infection, although antigens previously identified as candidate host-protective antigens (Bowtell, Mitchell, Anders, Lightowlers & Rickard, 1983) have yet to be identified in the expression library.

Conserved sequences flank variable tandem repeats in two S-antigen genes of Plasmodium falciparum.

We describe the isolation of two chromosomal DNA fragments from Plasmodium falciparum. These fragments encode the antigenically distinct S antigens of two different P. falciparum isolates, namely FC27 from Papua New Guinea and NF7 from Ghana. The complete nucleotide sequences of both fragments are presented. The fragments are homologous over most of their lengths, including the entire regions flanking the protein coding sequences. Whereas the N- and C-terminal portions of sequences encoding the S antigens are homologous, major portions of the coding sequences are not. The nonhomologous regions are comprised of tandemly repeated sequences, of 33 bp in FC27 and predominantly of 24 bp in NF7. The 33 bp tandem repeats encoded by the FC27 S-antigen gene could not be detected in the NF7 genome. Conversely, the 24 bp tandem repeats encoded by the NF7 S-antigen gene could not be detected in the FC27 genome. The pattern of sequence variation within the repeats of both genes suggests a mechanism for the generation of S-antigen diversity.

Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila.

The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans.

Many proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man. As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P. falciparum cDNA sequences, cloned in Escherichia coli. The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11. About 5% of the resulting clones expressed P. falciparum sequences as polypeptides fused to beta-galactosidase. We have identified many clones that express P. falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P. falciparum growth in vitro. The antigen-positive clones contain P. falciparum cDNA sequences, as determined by hybridization. Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P. falciparum. The cloned P. falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules.

Isolate-specific S-antigen of Plasmodium falciparum contains a repeated sequence of eleven amino acids.

Antibodies raised against a Plasmodium falciparum protein expressed in Escherichia coli reacted with a 220,000-molecular weight antigen of mature blood-stage parasites. The protein resembles the sporozoite surface antigen being composed of tandem repeats of 11 amino acids. However, it is isolate-specific and the encoding gene is not detectable in strains that do not express the protein.

Plasmodium falciparum: differentiation of isolates with DNA hybridization using antigen gene probes.

Chromosomal DNA was prepared from seven Plasmodium falciparum isolates that had been cultured in vitro and from a cloned P. falciparum line. The DNA was cleaved with restriction endonucleases, fractionated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized with a series of radioactively labeled DNA probes. The probes had been derived from cDNA clones encoding portions of P. falciparum antigens. Simple, reproducible band patterns that differed for many of the isolates were obtained. Parasite isolates collected from different continents could be readily distinguished, as could some but not all isolates collected from one restricted region of Papua New Guinea. Application of this technique for the identification and differentiation of parasite strains was explored. The patterns of hybridization observed were consistent with the proposition that blood stages of P. falciparum have a haploid genome.

Plasmodium falciparum complementary DNA clones expressed in Escherichia coli encode many distinct antigens.

A library of cDNA clones expressing antigens of the asexual blood-stages of Plasmodium falciparum (isolate FCQ27/PNG) was constructed in the bacteriophage vector gamma gt11-Amp3. Clones expressing P. falciparum antigens (as polypeptides fused to beta-galactosidase) were selected by their reactivity in an in situ colony immunoassay with affinity-purified malaria antibodies. A detailed analysis of 78 antigen-positive clones selected from approximately 10,000 recombinant clones has shown them to correspond to many different parasite antigens. cDNA hybridization studies on this array of 78 antigen-positive clones have so far identified 18 families of sibling clones with 22 clones as yet unassigned, the majority of which may represent additional unique sequences. Only about 20% of the clones synthesized abundant amounts of the malaria antigen/beta-galactosidase fused polypeptide but each multi-member family except one was represented by at least one clone producing a fused polypeptide in abundance. Antisera have been raised against cloned malaria antigens by immunizing mice and rabbits with bacterial lysates and purified fused polypeptides, respectively. These antisera have been used to characterize the antigens in P. falciparum that correspond to the various antigen-positive clones. The variety of distinct antigens recognized by these antisera confirms that the clone library contains coding sequences for many different antigens.

Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences.

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.

Plasmodium falciparum: identification and localization of a knob protein antigen expressed by a cDNA clone.

Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the FCQ27/PNG isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.

The ring-infected erythrocyte surface antigen (RESA) polypeptide of Plasmodium falciparum contains two separate blocks of tandem repeats encoding antigenic epitopes that are naturally immunogenic in man.

We showed previously that the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum contains a repetitive amino acid sequence. We have investigated here the sequence and antigenic relationships between RESA from FC27, a Papua New Guinea isolate, and from NF7, a Ghanaian isolate. The complete nucleotide sequences of eight different cDNA clones demonstrate that RESA from the two strains are closely homologous over the region that can be compared. A series of related eight, four and three amino acid repeats is located at the 3' end, forming the C terminus of RESA in FC27. RESA contains a second block of repeats based on an 11 amino acid sequence and separated from the 3' block by 381 amino acids in NF7. Antibodies from Papua New Guineans react with RESA from the African isolate, and vice-versa. Antigenic determinants that are naturally immunogenic in man are present in the 5' repeats of RESA, as well as in the 3' repeats, and antibodies that cross-react with both blocks of repeats were detected by reacting affinity-purified human antibodies with cloned subfragments of the cDNA clones, expressed in Escherichia coli.

The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli.

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.